Diagnostic preparation for the determination of serum bilirubin



United States Patent 1 2,737,501 DIAGNOSTIC PREPARATION FOR THE DETER- MINATION OF SERUM BILIRUBIN Leonard Sherman, Dearborn, Mich., assignor to Cambridge Chemical Products Company, Dearborn, Mich., a corporation of Michigan No Drawing. Continuation of application Serial No. 345,735, March 30, 1953. This application Ctctober 6, 1954, Serial No. 460,763

6 Claims. (Cl. 25Z408) This application is a continuation as to all common subject matter of my application Serial Number 345,735, filed March 30, 1953, now abandoned.

The invention relates to a diagnostic preparation, and, more particularly, to a stable composition for use in the determination of serum bilirubin, which is a liver function test.

A primary object is the preparation of a stable composition in tablet form to replace two solutions now required for this purpose, one of said solutions being only relatively stable, while the other is so unstable that it must be prepared immediately prior to each test or serum determination.

The prior art method is known as the Van Den Bergh Test or Ehrlichs Diazo Reagent Modified, and is fully described in Remingtons Practice of Pharmacy, pages 1142-43, 10th edition, Cook-Martin, 1951.

The present practice in serum bilirubin determination may be summarized as follows:

First, there is prepared what is termed a diazo reagent composed of diazo A solution and diazo B solution. Diazo A solution consists of 1% sulfanilic acid in 1.5% hydrochloric acid. This solution is, at best, stable for about one month. Diazo B solution consists of 0.5% sodium nitrite; it is very unstable and must be weighed out and freshly made prior to each test or determination. Diazo reagent composed of diazo A and diazo B, as above described, combines with bilirubin to give a reddish purple compound known as azobilin. The intensity of this color can be measured in a colorimeter for a determination of the bilirubin present in the serum.

It will now be appreciated that the making of a fresh batch of diazo B prior to each determination, and possibly also diazo A, consumes a great deal of time and opens the way for errors in measurement, etc. This is all obviated, according to the instant invention, by compounding a dry product composed of sodium sulfanilate, sodium nitrite, and a water-soluble binder, such as lactose, dextrose, dextrin, and the like.

Five parts of sodium sulfanilate are mixed with one part of sodium nitrite and suflicient quantity of a selected water-soluble binder whereby the dry product may be readily formed into tablets. It will be apparent that the quantity of binder used will vary with the character thereof, and the proportion thereof relative to the other ingredients is, obviously, not critical. Each tablet will contain 0.1 gram of sodium sulfanilate and 0.02 gram of sodium nitrite. Tablets thus made are found to be stable and keep indefinitely if preserved dry in a stoppered bottle.

It will be understood that the invention is not restricted to the use of sodium sulfanilate as one of the critical ingredients of my composition, but that any salt of sulfanilic acid, such as potassium or lithium sulfanilate, may be used instead.

Similarly, the sodium nitrite of my composition may be substituted by a nitrite such as potassium nitrite or lithium nitrite.

It will be also understood that the proportion of the active ingredients is the preferred one, but that it may be subjected to some variations without affecting the usefulness of the composition for the uses specified herein.

In the data appearing below, as a matter of convenience, the tablets of the instant invention have been designated as Bilitest tablets.

The determination of serum bilirubin (Evelyn and Malloy--modified) Filter: 540 (Green) Reagents:

Methyl alcohol, absolute, C. P. Diazo blank reagent, dilute 15 ml. of cone. HCl to 1 liter with distilled water. Bilitest tablets, dissolve one tablet in 25 ml. of diazo blank reagent. Bilirubin standard, a standard which corresponds to 5l0 mg. percent serum bilirubin is satisfactory. Procedure: Dilute 1.0 ml. of serum to 10.0 ml. with distilled Water. Prepare the following three colorimeter tubes:

Standard:

5.0 ml. bilirubin standard. 1.0 ml. diluted Bilitest. 4.0 ml. methyl alcohol. Blank:

5 .0 ml. methyl alcohol. 1.0 ml. diazo blank reagent. 4.0 ml. diluted serum. Unknown:

5.0 ml. methyl alcohol. 1.0 ml. diluted Bilitest. 4.0 ml. diluted serum. Mix all three tubes by gentle inversion and let stand 30 minutes. Read in colorimeter, subtracting the blank from the unknown.

Calculations Cone. of stand. Read. of stand. XRead' of unkown mg. percent of bilirubin in serum Cone. of stand. Read. of stand.

It will now be seen that I have attained the aims and purposes of the instant invention, and what I claim and desire to secure by Letters Patent is:

l. A tablet useful in the determination of serum bilirubin, consisting of five parts of sodium sulfanilate, one part of sodium nitrite, and a water-soluble binder.

2. A tablet useful in the determination of serum bilirubin, consisting of 0.1 gram of sodium sulfanilate and 0.02 gram of sodium nitrite.

3. A tablet useful in the determination of serum bilirubin, consisting of 0.1 gram of sodium sulfanilate, 0.02 gram of sodium nitrite, and a water-soluble binder.

4. The tablet of claim 3 in which said water-soluble binder is a member chosen from the group consisting of lactose, dextrose, and dextrin.

5. A dry preparation useful in the determination of serum bilirubin, consisting of an alkali metal salt of sulfanilic acid and sodium nitrite in proportions of five parts to one, in the order stated, and a Water-soluble binder.

6. A dry preparation for the determination of serum bilirubin, the preparation consisting of an alkali metal salt of sulfanilic acid and an alkali metal nitrite salt in proportion of approximately five parts of the first-named salt to one part of any of the nitrite salts, and a watersoluble binder.

=calibration factor Remingtons Practice of Pharmacy, pp. 1142-3, 10th ed., Cook-Martin, 1951. 

1. A TABLET USEFUL IN THE DETERMINATION OF SERUM BILIRUBIN, CONSISTING OF FIVE PART OF SODIUM SULFANILATE, ONE PART OF SODIUM NITRITE, AND A WATER-SOLUBLE BINDER. 